Compositions and methods for inhibiting &#34;stimulator of interferon gene&#34; -dependent signalling

ABSTRACT

The present invention provides cyclic-di-nucleotide (CDN) compounds that inhibit signaling at a recently discovered cytoplasmic receptor known as STING (Stimulator of Interferon Genes). In particular, the CDNs of the present invention are provided in the form of a composition comprising one or more cyclic purine dinucleotides which inhibit STING-dependent TBK1 activation and the resulting production of type I interferon.

The present application claims priority to U.S. Provisional Application61/825,009 filed May 18, 2013, and to U.S. Provisional Application61/902,125 filed Nov. 8, 2013, each of which is hereby incorporated byreference in its entirety.

BACKGROUND OF THE INVENTION

The following discussion of the background of the invention is merelyprovided to aid the reader in understanding the invention and is notadmitted to describe or constitute prior art to the present invention.

The human immune system may generally be divided into two arms, referredto as “innate immunity” and “adaptive immunity.” The innate arm of theimmune system is predominantly responsible for an initial inflammatoryresponse via a number of soluble factors, including the complementsystem and the chemokine/cytokine system; and a number of specializedcell types including mast cells, macrophages, dendritic cells (DCs), andnatural killer cells. In contrast, the adaptive immune arm involves adelayed and a longer lasting antibody response together with CD8+ andCD4+ T cell responses that play a critical role in immunological memoryagainst an antigen. A third arm of the immune system may be identifiedas involving γδ T cells and T cells with limited T cell receptorrepertoires such as NKT cells and MAIT cells.

For an effective immune response to an antigen, antigen presenting cells(APCs) must process and display the antigen in a proper MHC context to aT cell, which then will result in either T cell stimulation of cytotoxicand helper T cells. Following antigen presentation successfulinteraction of co-stimulatory molecules on both APCs and T cells mustoccur or activation will be aborted. GM-CSF and IL-12 serve as effectivepro-inflammatory molecules in many tumor models. For example, GM-CSFinduces myeloid precursor cells to proliferate and differentiate intodendritic cells (DCs) although additional signals are necessary toactivate their maturation to effective antigen-presenting cellsnecessary for activation of T cells. Barriers to effective immunetherapies include tolerance to the targeted antigen that can limitinduction of cytotoxic CD8 T cells of appropriate magnitude andfunction, poor trafficking of the generated T cells to sites ofmalignant cells, and poor persistence of the induced T cell response.

DCs that phagocytose tumor-cell debris process the material for majorhistocompatibility complex (MHC) presentation, upregulate expression ofcostimulatory molecules, and migrate to regional lymph nodes tostimulate tumor-specific lymphocytes. This pathway results in theproliferation and activation of CD4+ and CD8+ T cells that react totumor-associated antigens. Indeed, such cells can be detected frequentlyin the blood, lymphoid tissues, and malignant lesions of patients.

New insights into the mechanisms underlying immune-evasion, togetherwith combination treatment regimens that potentiate the potency oftherapeutic vaccination—either directly or indirectly—throughcombination with immune checkpoint inhibitors or other therapies, haveserved as a basis for the development of vaccines that induce effectiveantitumor immunity. The CDNs cyclic-di-AMP (produced by Listeriamonocytogenes) and its analog cyclic-di-GMP (produced by Legionellapneumophila) are recognized by the host cell as a PAMP (PathogenAssociated Molecular Pattern), which bind to the PRR (PathogenRecognition Receptor) known as STING. STING is an adaptor protein in thecytoplasm of host mammalian cells which activates the TANK bindingkinase (TBK1)-IRF3 signaling axis, resulting in the induction of IFN-βand other IRF-3 dependent gene products that strongly activate innateimmunity. It is now recognized that STING is a component of the hostcytosolic surveillance pathway, that senses infection with intracellularpathogens and in response induces the production of IFN-β, leading tothe development of an adaptive protective pathogen-specific immuneresponse consisting of both antigen-specific CD4 and CD8 T cells as wellas pathogen-specific antibodies. Examples of cyclic purine dinucleotidesare described in some detail in, e.g., U.S. Pat. Nos. 7,709,458 and7,592,326; WO2007/054279; and Yan et al., Bioorg. Med. Chem Lett. 18:5631 (2008), each of which is hereby incorporated by reference.

There remains a need for improved compositions and methods forimmunologic strategies to treating diseases such as cancer that can berefractory to traditional therapeutic approaches.

SUMMARY OF THE INVENTION

It is an object of the present invention to provide combinationtherapies for the treatment of cancer.

In a first aspect, the present invention provides compositionscomprising:

one or more cyclic purine dinucleotides (“CDNs”) which that inhibitSTimulator of INTerferon Gene (“STING”)-dependent type I interferonproduction. As described hereinafter, a number of CDNs find use in thepresent invention. Preferred cyclic purine dinucleotides include, butare not limited to, one or more of c-di-AMP, c-di-GMP, c-di-IMP,c-AMP-GMP, c-AMP-IMP, c-GMP-IMP, and analogs thereof. This list is notmeant to be limiting.

The general structure of a cyclic purine dinucleotide according to thepresent invention is as follows:

where each R1 and R2 is a purine, and the structure

is intended to reflect that the phosphate linkage may be to either the2′ or 3′ position on the ribose, and the other of the 2′ or 3′ positionwhich is not participating in the cyclic linkage is an —OH. Thus, thepresent invention contemplates 2′,5′,2′,5′ CDNs, 2′,5′,3′,5′ CDNs, and3′,5′,3′,5′ CDNs. BY way of example, c-di-GMP having 3′-5′ linkagesrefers to the molecule indicated above where each of R1 and R2 areguanine, and each phosphate linkage is 3′-to-5′.

For purposes of the present invention, this general structure is furthermodified to introduce substituents which confer the ability to inhibitSTING-dependent signaling, and thereby inhibit STING-dependent type Iinterferon production. By way of example, the present invention providescompositions comprising the following compounds:

wherein each X is independently O or S, and R3 and R4 are eachindependently H or an optionally substituted straight chain alkyl offrom 1 to 18 carbons and from 0 to 3 heteroatoms, an optionallysubstituted alkenyl of from 1-9 carbons, an optionally substitutedalkynyl of from 1-9 carbons, or an optionally substituted aryl, whereinsubstitution(s), when present, may be independently selected from thegroup consisting of C₁₋₆ alkyl straight or branched chain, benzyl,halogen, trihalomethyl, C₁₋₆ alkoxy, —NO₂, —NH₂, —OH, ═O, —COOR′ whereR′ is H or lower alkyl, —CH₂OH, and —CONH₂, wherein R3 and R4 are notboth H.

In preferred embodiments, one or both of R3 and R4 are independently anunsubstituted straight chain alkyl of from 1 to 18 carbons, anunsubstituted alkenyl of from 1-9 carbons, an unsubstituted alkynyl offrom 1-9 carbons, or an unsubstituted aryl. In certain embodiments, oneor both of R3 and R4 are allyl, propargyl, homoallyl, homopropargyl,methyl, ethyl, propyl, isopropyl, isobutyl, cyclopropylmethyl, orbenzyl, either substituted or unsubstituted. In certain embodiments,one, but not both, or R3 and R4 provide a prodrug leaving group such asan aliphatic ester which is removed by cellular esterases.

In certain embodiments, each X is S. In preferred embodiments when eachX is S, the compositions comprise one or more substantially pure Sp,Sp,Rp,Rp, Sp,Rp, or Rp,Sp stereoisomers.

In certain embodiments, each of R1 and R2 are independently selectedfrom the group consisting of adenine, guanine, inosine, and xanthine oranalogs thereof. Preferably, each of R1 and R2 are independently adenineor guanine.

As described hereinafter, a cyclic purine dinucleotide compositionaccording to the present invention can inhibit STING-dependent type Iinterferon production at least 2-fold, and more preferably 5-fold or10-fold, or more, as compared to c-di-GMP having 3′-5′ linkages.

The compositions of the present invention may be administered toindividuals in need thereof by a variety of parenteral and nonparenteralroutes in formulations containing pharmaceutically acceptable carriers,adjuvants and vehicles. Preferred routes are parenteral, and includebut, are not limited to, one or more of subcutaneous, intravenous,intramuscular, intraarterial, intradermal, intrathecal and epiduraladministrations. Particularly preferred is administration bysubcutaneous administration. Preferred pharmaceutical compositions areformulated as aqueous, liposomal, or oil-in-water emulsions. Exemplarycompositions are described hereinafter.

In related aspects, the present invention relates to methods ofinhibiting or moderating an immune response in an individual, comprisingadministering a composition according to the present invention to anindividual in need thereof. In other related aspects, the presentinvention relates to methods of inhibiting or moderating type Iinterferon production in an individual, comprising administering acomposition according to the present invention to an individual in needthereof. Examples of autoimmune diseases which may be treated using thecompositions of the present invention include, but are not limited to,alopecia areata, autoimmune hemolytic anemia, autoimmune hepatitis,dermatomyositis, diabetes (type 1), autoimmune juvenile idiopathicarthritis, glomerulonephritis, Graves' disease, Guillain-Barré syndrome,idiopathic thrombocytopenic purpura, lupus, myasthenia gravis, someforms of myocarditis, multiple sclerosis, pemphigus/pemphigoid,pernicious anemia, polyarteritis nodosa, polymyositis, primary biliarycirrhosis, psoriasis, rheumatoid arthritis, scleroderma/systemicsclerosis, Sjögren's syndrome, systemic lupus erythematosus, some formsof thyroiditis, some forms of uveitis, vitiligo, and granulomatosis withpolyangiitis (Wegener's).

In other embodiments, the methods described herein can compriseadministering to the mammal an effective amount of the substantiallypure CDNs of the present invention for the treatment of disorders inwhich shifting of Th1 to Th2 immunity confers clinical benefit.Cell-mediated immunity (CMI) is associated with TH1 CD4+ T lymphocytesproducing cytokines IL-2, interferon (IFN)-γ and tumor necrosis factor(TNF)-α. In contrast, humoral immunity is associated with TH2 CD4+ Tlymphocytes producing IL-4, IL-6 and IL-10 Immune deviation towards TH1responses typically produces activation of cytotoxic T-cell lymphocytes(CTL), natural killer (NK) cells, macrophages and monocytes. Generally,Th1 responses are more effective against intracellular pathogens(viruses and bacteria that are inside host cells) and tumors, while Th2responses are more effective against extracellular bacteria, parasitesincluding helminths and toxins. Type I interferons (IFNs-I) are believedto mediate the lethal effects of endotoxemia and sepsis, and so themethods and compositions of the present invention can find use in thetreatment of sepsis. In addition, the activation of innate immunity isexpected to normalize the T-helper type 1 and 2 (Th1/Th2) immune systembalance and to suppress the excessive reaction of Th2 type responsesthat cause immunoglobulin (Ig) E-dependent allergies and allergicasthma.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 depicts cyclic purine dinucleotide (“CDN”)-mediated signaling. ACDN (e.g., c-di-AMP or c-di-GMP) induces production of IFN-β by bindingto the cytosolic adaptor protein STING (Stimulator of Interferon Genes),and inducing signaling through the TBK-1/IRF-3 pathway, resulting inboth autocrine and paracrine activation of DCs through binding to theIFN receptor and subsequent signaling.

FIG. 2 depicts a synthesis scheme Synthesis of2′-O-propargyl-cyclic-A(2′,5′)pA(3′,5′)p (2′-O-propargyl-ML-CDA).

FIG. 3A depicts ¹H NMR analytical results for 2′-O-propargyl-ML-CDA(compound 8).

FIG. 3B depicts ³¹P NMR analytical results for 2′-O-propargyl-ML-CDA(compound 8).

FIG. 3C depicts COSY (2.5-6.5 ppm—¹H axis) analytical results for2′-O-propargyl-ML-CDA (compound 8).

FIG. 3D depicts HMBC (3.5-6.5 ppm—¹H axis) analytical results for2′-O-propargyl-ML-CDA (compound 8).

FIG. 3E depicts analytical HPLC (2-20% ACN/10 mM TEAA buffer—20 min)analytical results for 2′-O-propargyl-ML-CDA (compound 8).

FIG. 4 depicts c-[G(2′,5′)pG(3′,5′)p] and dithio ribose O-substitutedderivatives.

FIG. 5 depicts c-[A(2′,5′)pA(3′,5′)p] and dithio ribose O-substitutedderivatives.

FIG. 6 depicts c-[G(2′,5′)pA(3′,5′)p] and dithio ribose O-substitutedderivatives.

FIG. 7 depicts2′-O-propargyl-cyclic-[A(2′,5′)pA(3′,5′)p](2′-O-propargyl-ML-CDA)inhibition of STING-dependent activation of type I interferon.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to the use of novel cyclic-di-nucleotide(CDN) compounds that inhibit signaling at a recently discoveredcytoplasmic receptor known as STING (Stimulator of Interferon Genes). Inparticular, the CDNs of the present invention are provided in the formof a composition comprising one or more cyclic purine dinucleotideswhich inhibit STING-dependent TBK1 activation and the resultingproduction of type I interferon.

The CDNs cyclic-di-AMP (produced by Listeria monocytogenes) and itsanalog cyclic-di-GMP (produced by Legionella pneumophila) are recognizedby the host cell as a PAMP (Pathogen Associated Molecular Pattern),which bind to the PRR (Pathogen Recognition Receptor) known as STING.STING is an adaptor protein in the cytoplasm of host mammalian cellswhich activates the TANK binding kinase (TBK1)-IRF3 signaling axis,resulting in the induction of IFN-γ and other IRF-3 dependent geneproducts that strongly activate innate immunity. It is now recognizedthat STING is a component of the host cytosolic surveillance pathway,that senses infection with intracellular pathogens and in responseinduces the production of IFN, leading to the development of an adaptiveprotective pathogen-specific immune response consisting of bothantigen-specific CD4 and CD8 T cells as well as pathogen-specificantibodies.

In the case of autoimmune diseases, inhibitors of this pathway canprovide a novel therapeutic route which has not been previouslyexploited.

DEFINITIONS

“Administration” as it is used herein with regard to a human, mammal,mammalian subject, animal, veterinary subject, placebo subject, researchsubject, experimental subject, cell, tissue, organ, or biological fluid,refers without limitation to contact of an exogenous ligand, reagent,placebo, small molecule, pharmaceutical agent, therapeutic agent,diagnostic agent, or composition to the subject, cell, tissue, organ, orbiological fluid, and the like. “Administration” can refer, e.g., totherapeutic, pharmacokinetic, diagnostic, research, placebo, andexperimental methods. Treatment of a cell encompasses contact of areagent to the cell, as well as contact of a reagent to a fluid, wherethe fluid is in contact with the cell. “Administration” also encompassesin vitro and ex vivo treatments, e.g., of a cell, by a reagent,diagnostic, binding composition, or by another cell. By “administeredtogether” it is not meant to be implied that two or more agents beadministered as a single composition. Although administration as asingle composition is contemplated by the present invention, such agentsmay be delivered to a single subject as separate administrations, whichmay be at the same or different time, and which may be by the same routeor different routes of administration.

An “agonist,” as it relates to a ligand and receptor, comprises amolecule, combination of molecules, a complex, or a combination ofreagents, that stimulates the receptor. For example, an agonist ofgranulocyte-macrophage colony stimulating factor (GM-CSF) can encompassGM-CSF, a mutein or derivative of GM-CSF, a peptide mimetic of GM-CSF, asmall molecule that mimics the biological function of GM-CSF, or anantibody that stimulates GM-CSF receptor.

An “antagonist,” as it relates to a ligand and receptor, comprises amolecule, combination of molecules, or a complex, that inhibits,counteracts, downregulates, and/or desensitizes the receptor.“Antagonist” encompasses any reagent that inhibits a constitutiveactivity of the receptor. A constitutive activity is one that ismanifest in the absence of a ligand/receptor interaction. “Antagonist”also encompasses any reagent that inhibits or prevents a stimulated (orregulated) activity of a receptor. By way of example, an antagonist ofGM-CSF receptor includes, without implying any limitation, an antibodythat binds to the ligand (GM-CSF) and prevents it from binding to thereceptor, or an antibody that binds to the receptor and prevents theligand from binding to the receptor, or where the antibody locks thereceptor in an inactive conformation.

By “substantially purified” with regard to CDNs of the invention ismeant that a specified species accounts for at least 50%, more oftenaccounts for at least 60%, typically accounts for at least 70%, moretypically accounts for at least 75%, most typically accounts for atleast 80%, usually accounts for at least 85%, more usually accounts forat least 90%, most usually accounts for at least 95%, and conventionallyaccounts for at least 98% by weight, or greater, of the CDN activitypresent in a composition. The weights of water, buffers, salts,detergents, reductants, protease inhibitors, stabilizers (including anadded protein such as albumin), and excipients are generally not used inthe determination of purity.

“Specifically” or “selectively” binds, when referring to aligand/receptor, nucleic acid/complementary nucleic acid,antibody/antigen, or other binding pair (e.g., a cytokine to a cytokinereceptor) (each generally referred to herein as a “target biomolecule”or a “target”) indicates a binding reaction which is related to thepresence of the target in a heterogeneous population of proteins andother biologics. Specific binding can mean, e.g., that the bindingcompound, nucleic acid ligand, antibody, or binding composition derivedfrom the antigen-binding site of an antibody, of the contemplated methodbinds to its target with an affinity that is often at least 25% greater,more often at least 50% greater, most often at least 100% (2-fold)greater, normally at least ten times greater, more normally at least20-times greater, and most normally at least 100-times greater than theaffinity with a non-target molecule.

“Ligand” refers to a small molecule, nucleic acid, peptide, polypeptide,saccharide, polysaccharide, glycan, glycoprotein, glycolipid, orcombinations thereof that binds to a target biomolecule. While suchligands may be agonists or antagonists of a receptor, a ligand alsoencompasses a binding agent that is not an agonist or antagonist, andhas no agonist or antagonist properties. Specific binding of a ligandfor its cognate target is often expressed in terms of an “Affinity.” Inpreferred embodiments, the ligands of the present invention bind withaffinities of between about 10⁴ M⁻¹ and about 10⁸ M⁻¹. Affinity iscalculated as K_(a)=k_(off)/k_(on) (k_(off) is the dissociation rateconstant, K_(on) is the association rate constant and K_(d) is theequilibrium constant).

Affinity can be determined at equilibrium by measuring the fractionbound (r) of labeled ligand at various concentrations (c). The data aregraphed using the Scatchard equation: r/c=K(n−r): where r=moles of boundligand/mole of receptor at equilibrium; c=free ligand concentration atequilibrium; K=equilibrium association constant; and n=number of ligandbinding sites per receptor molecule. By graphical analysis, r/c isplotted on the Y-axis versus r on the X-axis, thus producing a Scatchardplot. Affinity measurement by Scatchard analysis is well known in theart. See, e.g., van Erp et al., J. Immunoassay 12: 425-43, 1991; Nelsonand Griswold, Comput. Methods Programs Biomed. 27: 65-8, 1988. In analternative, affinity can be measured by isothermal titrationcalorimetry (ITC). In a typical ITC experiment, a solution of ligand istitrated into a solution of its cognate target. The heat released upontheir interaction (ΔH) is monitored over time. As successive amounts ofthe ligand are titrated into the ITC cell, the quantity of heat absorbedor released is in direct proportion to the amount of binding. As thesystem reaches saturation, the heat signal diminishes until only heatsof dilution are observed. A binding curve is then obtained from a plotof the heats from each injection against the ratio of ligand and bindingpartner in the cell. The binding curve is analyzed with the appropriatebinding model to determine K_(B), n and ΔH. Note that K_(B)=1/K_(d).

The term “subject” as used herein refers to a human or non-humanorganism. Thus, the methods and compositions described herein areapplicable to both human and veterinary disease. In certain embodiments,subjects are “patients,” i.e., living humans that are receiving medicalcare for a disease or condition. This includes persons with no definedillness who are being investigated for signs of pathology. Preferred aresubjects who have an existing diagnosis of a particular cancer which isbeing targeted by the compositions and methods of the present invention.Preferred cancers for treatment with the compositions described hereininclude, but are not limited to prostate cancer, renal carcinoma,melanoma, pancreatic cancer, cervical cancer, ovarian cancer, coloncancer, head & neck cancer, lung cancer and breast cancer.

“Therapeutically effective amount” is defined as an amount of a reagentor pharmaceutical composition that is sufficient to show a patientbenefit, i.e., to cause a decrease, prevention, or amelioration of thesymptoms of the condition being treated. When the agent orpharmaceutical composition comprises a diagnostic agent, a“diagnostically effective amount” is defined as an amount that issufficient to produce a signal, image, or other diagnostic parameter.Effective amounts of the pharmaceutical formulation will vary accordingto factors such as the degree of susceptibility of the individual, theage, gender, and weight of the individual, and idiosyncratic responsesof the individual. “Effective amount” encompasses, without limitation,an amount that can ameliorate, reverse, mitigate, prevent, or diagnose asymptom or sign of a medical condition or disorder or a causativeprocess thereof. Unless dictated otherwise, explicitly or by context, an“effective amount” is not limited to a minimal amount sufficient toameliorate a condition.

“Treatment” or “treating” (with respect to a condition or a disease) isan approach for obtaining beneficial or desired results including andpreferably clinical results. For purposes of this invention, beneficialor desired results with respect to a disease include, but are notlimited to, one or more of the following: preventing a disease,improving a condition associated with a disease, curing a disease,lessening severity of a disease, delaying progression of a disease,alleviating one or more symptoms associated with a disease, increasingthe quality of life of one suffering from a disease, and/or prolongingsurvival. Likewise, for purposes of this invention, beneficial ordesired results with respect to a condition include, but are not limitedto, one or more of the following: preventing a condition, improving acondition, curing a condition, lessening severity of a condition,delaying progression of a condition, alleviating one or more symptomsassociated with a condition, increasing the quality of life of onesuffering from a condition, and/or prolonging survival. For instance, inembodiments where the compositions described herein are used fortreatment of cancer, the beneficial or desired results include, but arenot limited to, one or more of the following: reducing the proliferationof (or destroying) neoplastic or cancerous cells, reducing metastasis ofneoplastic cells found in cancers, shrinking the size of a tumor,decreasing symptoms resulting from the cancer, increasing the quality oflife of those suffering from the cancer, decreasing the dose of othermedications required to treat the disease, delaying the progression ofthe cancer, and/or prolonging survival of patients having cancer.Depending on the context, “treatment” of a subject can imply that thesubject is in need of treatment, e.g., in the situation where thesubject comprises a disorder expected to be ameliorated byadministration of a reagent.

The term “antibody” as used herein refers to a peptide or polypeptidederived from, modeled after or substantially encoded by animmunoglobulin gene or immunoglobulin genes, or fragments thereof,capable of specifically binding an antigen or epitope. See, e.g.Fundamental Immunology, 3rd Edition, W. E. Paul, ed., Raven Press, N.Y.(1993); Wilson (1994; J. Immunol. Methods 175:267-273; Yarmush (1992) J.Biochem. Biophys. Methods 25:85-97. The term antibody includesantigen-binding portions, i.e., “antigen binding sites,” (e.g.,fragments, subsequences, complementarity determining regions (CDRs))that retain capacity to bind antigen, including (i) a Fab fragment, amonovalent fragment consisting of the VL, VH, CL and CH1 domains; (ii) aF(ab′)2 fragment, a bivalent fragment comprising two Fab fragmentslinked by a disulfide bridge at the hinge region; (iii) a Fd fragmentconsisting of the VH and CH1 domains; (iv) a Fv fragment consisting ofthe VL and VH domains of a single arm of an antibody, (v) a dAb fragment(Ward et al., (1989) Nature 341:544-546), which consists of a VH domain;and (vi) an isolated complementarity determining region (CDR). Singlechain antibodies are also included by reference in the term “antibody.”

Cyclic Purine Dinucleotides

Prokaryotic as well as eukaryotic cells use various small molecules forcell signaling and intra- and intercellular communication. Cyclicnucleotides like cGMP, cAMP, etc. are known to have regulatory andinitiating activity in pro- and eukaryotic cells. Unlike eukaryoticcells, prokaryotic cells also use cyclic purine dinucleotides asregulatory molecules. In prokaryotes, the condensation of two GTPmolecules is catalyst by the enzyme diguanylate cyclase (DGC) to givec-diGMP, which represents an important regulator in bacteria.

Recent work suggests that cyclic diGMP or analogs thereof can alsostimulate or enhance immune or inflammatory response in a patient or canenhance the immune response to a vaccine by serving as an adjuvant inmammals. Cytosolic detection of pathogen-derived DNA requires signalingthrough TANK binding kinase 1 (TBK1) and its downstream transcriptionfactor, IFN-regulatory factor 3 (IRF3). A transmembrane protein calledSTING (stimulator of IFN genes; also known as MITA, ERIS, MPYS andTMEM173) functions as the signaling receptor for these cyclic purinedinucleotides, causing stimulation of the TBK1-IRF3 signalling axis anda STING-dependent type I interferon response. See, e.g., FIG. 1.Burdette et al., Nature 478: 515-18, 2011 demonstrated that STING bindsdirectly to cyclic diguanylate monophosphate, but not to other unrelatednucleotides or nucleic acids.

Cyclic purine dinucleotides for use as precursors to derive the CDNs ofthe present invention are described in some detail in, e.g., Gao et al.,Cell (2013) 153: doi: 10.1016/j.cell.2013.04.046; U.S. Pat. Nos.7,709,458 and 7,592,326; WO2007/054279; and Yan et al., Bioorg. Med.Chem Lett. 18: 5631 (2008), each of which is hereby incorporated byreference. These CDNs may be modified using standard organic chemistrytechniques in order to produce the CDNs of the present invention.

Preferred purines include, but are not limited to, adenine, guanine,inosine, hypoxanthine, xanthine, isoguanine, etc. The CDNs of thepresent invention are preferably phosphorothioate analogues, and mostpreferably substantially pure Sp,Sp, Rp,Rp, SpRp, or Rp,Sp stereoisomersthereof.

As denoted in the structures, each ribose comprises a 2′ or 3′ hydroxylwhich may be substituted. As described hereinafter, the CDNs of thepresent invention can comprise a substitution at one or both of these 2′or 3′ hydroxyls (which is not part of the cyclic linkage) which providea blocking moiety that is not removed as a prodrug leaving group. Suchsubstitutions include, but are not limited to, O-methyl, O-ethyl,O-propyl, O-isopropyl, O-benzyl, O-methoxyethyl, O-aminoethyl,O-propargyl, O-allyl, etc. This list is not meant to be limiting. Theterm “prodrug” as used herein refers to a modification of contemplatedcompounds, wherein the modified compound exhibits less pharmacologicalactivity (as compared to the modified compound) and wherein the modifiedcompound is converted within the body (e.g., in a target cell or targetorgan) back into the unmodified form through enzymatic or non-enzymaticreactions. In certain embodiments, the hydroxyl on one ribose comprisesa prodrug leaving group. Prodrugs can modify the physicochemical,biopharmaceutic, and pharmacokinetic properties of drugs. Traditionalprodrugs are classified as drugs that are activated by undergoingtransformation in vivo to form the active drug. Reasons for prodrugdevelopment are typically poor aqueous solubility, chemical instability,low oral bioavailability, lack of blood brain barrier penetration, andhigh first pass metabolism associated with the parent drug. Suitableprodrug moieties are described in, for example, “Prodrugs and TargetedDelivery,” J. Rautico, Ed., John Wiley & Sons, 2011.

Preferred cyclic purine dinucleotides are phosphorothioate analogues,referred to herein as “thiophosphates”. Phosphorothioates are a variantof normal nucleotides in which one of the nonbridging oxygens isreplaced by a sulfur. The sulfurization of the internucleotide bonddramatically reduces the action of endo- and exonucleases, including 5′to 3′ and 3′ to 5′ DNA POL 1 exonuclease, nucleases S1 and P1, RNases,serum nucleases and snake venom phosphodiesterase. In addition, thepotential for crossing the lipid bilayer increases.

A phosphorothioate linkage in inherently chiral. The skilled artisanwill recognize that the phosphates in this structure may each exist in Ror S forms. Thus, Rp,Rp, Sp,Sp, Sp,Rp, and Rp,Sp forms are possible.

As noted above, cyclic purine dinucleotides of the present inventioncomprise 2′-O- and 3′-O- substituent forms of CDNs, and in particularCDN thiophosphates. Additional stability and bioavailability can beprovided by the substitution of the 2′-OH of the ribose moiety.Substituent groups amenable herein include without limitation, halogen,hydroxyl, alkyl, alkenyl, alkynyl, acyl (—C(O)R_(aa)), carboxyl(—C(O)0-R_(aa)), aliphatic groups, alicyclic groups, alkoxy, substitutedoxy (-0-R_(aa)), aryl, aralkyl, heterocyclic radical, heteroaryl,heteroarylalkyl, amino (—N(R_(bb))(R_(cc))), imino (═NR_(bb)), amido(—C(O)N(R_(bb))(Rc_(c)) or —N(R_(bb))C(O)R_(aa)), azido (—N₃), nitro(—N0₂), cyano (—CN), carbamido (—OC(O)N(R_(bb))(R_(cc)) or—N(R_(bb))C(O)OR_(aa)), ureido (—N(R_(bb))C(O)—N(Rbb)(Rcc)), thioureido(—N(R_(bb))C(S)N(R_(bb))(R_(cc))), guanidinyl(—N(R_(bb))C(═NR_(bb))N(R_(bb))(R_(cc))), amidinyl(—C(═NR_(bb))N(R_(bb))(Rc_(c)) or —N(R_(bb))C(═NR_(bb))(R_(aa))), thiol(—SR_(bb)), sulfinyl (—S(O)R_(bb)), sulfonyl (—S(O)₂R_(b)) andsulfonamidyl (—S(O)₂N(R_(bb))(Rc_(c)) or —N(R_(bb))S(O)₂R_(bb)). Whereineach R_(aa), R_(bb) and R_(cC) is, independently, H, an optionallylinked chemical functional group or a further substituent group with apreferred list including without limitation, H, alkyl, alkenyl, alkynyl,aliphatic, alkoxy, acyl, aryl, aralkyl, heteroaryl, alicyclic,heterocyclic and heteroarylalkyl. Selected substituents within thecompounds described herein are present to a recursive degree.

The term “alkyl,” as used herein, refers to a saturated straight orbranched hydrocarbon radical containing up to twenty four carbon atoms.Examples of alkyl groups include without limitation, methyl, ethyl,propyl, butyl, isopropyl, n-hexyl, octyl, decyl, dodecyl and the like.Alkyl groups typically include from 1 to about 24 carbon atoms, moretypically from 1 to about 12 carbon atoms with from 1 to about 6 carbonatoms being more preferred. The term “lower alkyl” as used hereinincludes from 1 to about 6 carbon atoms. Alkyl groups as used herein mayoptionally include one or more further substituent groups.

The term “alkenyl,” as used herein, refers to a straight or branchedhydrocarbon chain radical containing up to twenty four carbon atoms andhaving at least one carbon-carbon double bond. Examples of alkenylgroups include without limitation, ethenyl, propenyl, butenyl,1-methyl-2-buten-1-yl, dienes such as 1,3-butadiene and the like.Alkenyl groups typically include from 2 to about 24 carbon atoms, moretypically from 2 to about 12 carbon atoms with from 2 to about 6 carbonatoms being more preferred. Alkenyl groups as used herein may optionallyinclude one or more further substituent groups.

The term “alkynyl,” as used herein, refers to a straight or branchedhydrocarbon radical containing up to twenty four carbon atoms and havingat least one carbon-carbon triple bond. Examples of alkynyl groupsinclude, without limitation, ethynyl, 1-propynyl, 1-butynyl, and thelike. Alkynyl groups typically include from 2 to about 24 carbon atoms,more typically from 2 to about 12 carbon atoms with from 2 to about 6carbon atoms being more preferred. Alkynyl groups as used herein mayoptionally include one or more further substituent groups.

The term “acyl,” as used herein, refers to a radical formed by removalof a hydroxyl group from an organic acid and has the general Formula—C(O)—X where X is typically aliphatic, alicyclic or aromatic. Examplesinclude aliphatic carbonyls, aromatic carbonyls, aliphatic sulfonyls,aromatic sulfinyls, aliphatic sulfinyls, aromatic phosphates, aliphaticphosphates and the like. Acyl groups as used herein may optionallyinclude further substituent groups.

The term “alicyclic” refers to a cyclic ring system wherein the ring isaliphatic. The ring system can comprise one or more rings wherein atleast one ring is aliphatic. Preferred alicyclics include rings havingfrom about 5 to about 9 carbon atoms in the ring. Alicyclic as usedherein may optionally include further substituent groups.

The term “aliphatic,” as used herein, refers to a straight or branchedhydrocarbon radical containing up to twenty four carbon atoms whereinthe saturation between any two carbon atoms is a single, double ortriple bond. An aliphatic group preferably contains from 1 to about 24carbon atoms, more typically from 1 to about 12 carbon atoms with from 1to about 6 carbon atoms being more preferred. The straight or branchedchain of an aliphatic group may be interrupted with one or moreheteroatoms that include nitrogen, oxygen, sulfur and phosphorus. Suchaliphatic groups interrupted by heteroatoms include without limitation,polyalkoxys, such as polyalkylene glycols, polyamines, and polyimines.Aliphatic groups as used herein may optionally include furthersubstituent groups.

The term “alkoxy,” as used herein, refers to a radical formed between analkyl group and an oxygen atom wherein the oxygen atom is used to attachthe alkoxy group to a parent molecule. Examples of alkoxy groups includewithout limitation, methoxy, ethoxy, propoxy, isopropoxy, n-butoxy,sec-butoxy, tert-butoxy, n-pentoxy, neopentoxy, n-hexoxy and the like.Alkoxy groups as used herein may optionally include further substituentgroups.

The term “aminoalkyl” as used herein, refers to an amino substitutedC\-Cn alkyl radical. The alkyl portion of the radical forms a covalentbond with a parent molecule. The amino group can be located at anyposition and the aminoalkyl group can be substituted with a furthersubstituent group at the alkyl and/or amino portions.

The terms “aralkyl” and “arylalkyl,” as used herein, refer to anaromatic group that is covalently linked to a C\-Cn alkyl radical. Thealkyl radical portion of the resulting aralkyl (or arylalkyl) groupforms a covalent bond with a parent molecule. Examples include withoutlimitation, benzyl, phenethyl and the like. Aralkyl groups as usedherein may optionally include further substituent groups attached to thealkyl, the aryl or both groups that form the radical group.

The terms “aryl” and “aromatic,” as used herein, refer to a mono- orpolycyclic carbocyclic ring system radicals having one or more aromaticrings. Examples of aryl groups include without limitation, phenyl,naphthyl, tetrahydronaphthyl, indanyl, idenyl and the like. Preferredaryl ring systems have from about 5 to about 20 carbon atoms in one ormore rings. Aryl groups as used herein may optionally include furthersubstituent groups.

The terms “halo” and “halogen,” as used herein, refer to an atomselected from fluorine, chlorine, bromine and iodine.

The terms “heteroaryl,” and “heteroaromatic,” as used herein, refer to aradical comprising a mono- or poly-cyclic aromatic ring, ring system orfused ring system wherein at least one of the rings is aromatic andincludes one or more heteroatoms. Heteroaryl is also meant to includefused ring systems including systems where one or more of the fusedrings contain no heteroatoms. Heteroaryl groups typically include onering atom selected from sulfur, nitrogen or oxygen. Examples ofheteroaryl groups include without limitation, pyridinyl, pyrazinyl,pyrimidinyl, pyrrolyl, pyrazolyl, imidazolyl, thiazolyl, oxazolyl,isooxazolyl, thiadiazolyl, oxadiazolyl, thiophenyl, furanyl, quinolinyl,isoquinolinyl, benzimidazolyl, benzooxazolyl, quinoxalinyl and the like.Heteroaryl radicals can be attached to a parent molecule directly orthrough a linking moiety such as an aliphatic group or hetero atom.Heteroaryl groups as used herein may optionally include furthersubstituent groups.

The term “heteroarylalkyl,” as used herein, refers to a heteroaryl groupas previously defined that further includes a covalently attached C₁-C₁₂alkyl radical. The alkyl radical portion of the resultingheteroarylalkyl group is capable of forming a covalent bond with aparent molecule. Examples include without limitation, pyridinylmethyl,pyrimidinylethyl, napthyridinylpropyl and the like. Heteroarylalkylgroups as used herein may optionally include further substituent groupson one or both of the heteroaryl or alkyl portions.

The following terms are defined as follows:

allyl —CH2CH═CH2,propargyl —CH2C≡CH,homoallyl —CH2CH2CH═CH2, andhomopropargyl —CH2CH2C≡CH.

As noted above, preferred cyclic purine dinucleotides also includeprodrug forms of CDNs, and in particular CDN thiophosphates. Prodrugscan modify the physicochemical, biopharmaceutic, and pharmacokineticproperties of drugs. Traditional prodrugs are classified as drugs thatare activated by undergoing transformation in vivo to form the activedrug. Reasons for prodrug development are typically poor aqueoussolubility, chemical instability, low oral bioavailability, lack ofblood brain barrier penetration, and high first pass metabolismassociated with the parent drug. Suitable prodrug moieties are describedin, for example, “Prodrugs and Targeted Delivery,” J. Rautico, Ed., JohnWiley & Sons, 2011.

The term “substantially pure” as used herein with regard to cyclicpurine dinucleotides refers to an Rp,Rp or Rp,Sp form which is at least75% pure relative to other possible stereochemistries at the chiralcenters indicated in the figure above. By way of example, a“substantially pure Rp,Rp c-di-GMP thiophosphate” would be at least 75%pure with regard to the Rp,Sp and Sp,Sp forms of c-di-GMP thiophosphate.In preferred embodiments, a substantially pure cyclic purinedinucleotide is at least 85% pure, at least 90% pure, at least 95% pure,at least 97% pure, and at least 99% pure. While a substantially purecyclic purine dinucleotide preparation of the invention is“stereochemically pure,” this is not meant to indicate that all CDNswithin the preparation having a particular stereochemistry at thesechiral centers are otherwise identical. For example, a substantiallypure cyclic purine dinucleotide preparation may contain a combination ofRp,Rp c-di-GMP thiophosphate and Rp,Rp c-di-AMP thiophosphate and stillbe a substantially pure cyclic purine dinucleotide preparation. Such apreparation may also include other components as described hereinafterthat are advantageous for patient treatment, provided that all CDNswithin the preparation having a particular stereochemistry at thesechiral centers.

The CDN compositions described herein can be administered to a host,either alone or in combination with a pharmaceutically acceptableexcipient, in an amount sufficient to modify an appropriate immuneresponse. The immune response can comprise, without limitation, specificimmune response, non-specific immune response, both specific andnon-specific response, innate response, primary immune response,adaptive immunity, secondary immune response, memory immune response,immune cell activation, immune cell proliferation, immune celldifferentiation, and cytokine expression. In certain embodiments, theCDN compositions are administered in conjunction with one or moreadditional compositions. The CDN compositions may be administeredbefore, after, and/or together with an additional therapeutic orprophylactic composition. Methods for co-administration with anadditional therapeutic agent are well known in the art (Hardman, et al.(eds.) (2001) Goodman and Gilman's The Pharmacological Basis ofTherapeutics, 10th ed., McGraw-Hill, New York, N.Y.; Poole and Peterson(eds.) (2001) Pharmacotherapeutics for Advanced Practice: A PracticalApproach, Lippincott, Williams & Wilkins, Phila., Pa.; Chabner and Longo(eds.) (2001) Cancer Chemotherapy and Biotherapy, Lippincott, Williams &Wilkins, Phila., Pa.). In certain embodiments the one or moretherapeutics is selected from anti-TNF agents (e.g., etanercept,infliximab), steroids, azathioprine, cyclosporine, methotrexate,abatacept, PDE4 inhibitors (e.g., roflumilast), etc.

Delivery Agents

Liposomes are vesicles formed from one (“unilamellar”) or more(“multilamellar”) layers of phospholipid. Because of the amphipathiccharacter of the phospholipid building blocks, liposomes typicallycomprise a hydrophilic layer presenting a hydrophilic external face andenclosing a hydrophilic core. The versatility of liposomes in theincorporation of hydrophilic/hydrophobic components, their non-toxicnature, biodegradability, biocompatibility, adjuvanticity, induction ofcellular immunity, property of sustained release and prompt uptake bymacrophages, makes them attractive candidates for the delivery ofantigens.

WO2010/104833, which is incorporated by reference herein in itsentirety, describes liposomal preparations which comprise:

-   -   a) an aqueous vehicle;    -   b) liposomes comprising    -   (i) dimyristoylphosphatidylcholine (“DMPC”),    -   (ii) dimyristoylphosphatidylglycerol (“DMPG”),        dimyristoyltrimethylammonium propane (“DMTAP”), or both DMPG and        DMTAP,    -   and    -   (iii) at least one sterol derivative; and    -   c) one or more immunogenic polypeptide(s) or carbohydrate(s)        covalently linked to between 1% and 100% of said at least one        sterol derivative.

Such liposomal formulations, referred to herein as VesiVax® (MolecularExpress, Inc.), with our without the “immunogenic polypeptide(s) orcarbohydrate(s)” referred to above, can contain one or more additionalcomponents such as peptidoglycan, lipopeptide, lipopolysaccharide,monophosphoryl lipid A, lipoteichoic acid, resiquimod, imiquimod,flagellin, oligonucleotides containing unmethylated CpG motifs,beta-galactosylceramide, muramyl dipeptide, all-trans retinoic acid,double-stranded viral RNA, heat shock proteins,dioctadecyldimethylammonium bromide, cationic surfactants, toll-likereceptor agonists, dimyristoyltrimethylammoniumpropane, and nod-likereceptor agonists. Advantageously, these liposomal formulations can beused to deliver one or more cyclic purine dinucleotides in accordancewith the present invention.

Moreover, while the liposomal formulations discussed above employ a“steroid derivative” as an anchor for attaching an immunogenicpolypeptide or carbohydrate to a liposome, the steroid may simply beprovided as an unconjugated steroid such as cholesterol.

Suitable methods for preparing liposomes from lipid mixtures are wellknown in the art. See, e.g., Basu & Basu, Liposome Methods and Protocols(Methods in Molecular Biology), Humana Press, 2002; Gregoriadis,Liposome Technology, 3^(rd) Edition, Informa HealthCare, 2006. Preferredmethods include extrusion, homogenization, and sonication methodsdescribed therein. An exemplary method for preparing liposomes for usein the present invention, which comprises drying a lipid mixture,followed by hydration in an aqueous vehicle and sonication to formliposomes, is described in WO2010/104833.

In certain embodiments, the liposomes are provided within a particularaverage size range. Liposome size can be selected, for example, byextrusion of an aqueous vehicle comprising liposomes through membraneshaving a preselected pore size and collecting the material flowingthrough the membrane. In preferred embodiments, the liposomes areselected to be substantially between 50 and 500 nm in diameter, morepreferably substantially between 50 and 200 nm in diameter, and mostpreferably substantially between 50 and 150 nm in diameter. The term“substantially” as used herein in this context means that at least 75%,more preferably 80%, and most preferably at least 90% of the liposomesare within the designated range.

Other lipid and lipid-like adjuvants which may find use in the presentinvention include oil-in-water (o/w) emulsions (see, e.g., Muderhwa etal., J. Pharmaceut. Sci. 88: 1332-9, 1999)), VesiVax® TLR (MolecularExpress, Inc.), digitonin (see, e.g., U.S. Pat. No. 5,698,432), andglucopyranosyl lipids (see, e.g., United States Patent Application20100310602).

Nanoparticles also represent drug delivery systems suitable for mostadministration routes. Over the years, a variety of natural andsynthetic polymers have been explored for the preparation ofnanoparticles, of which Poly(lactic acid) (PLA), Poly(glycolic acid)(PGA), and their copolymers (PLGA) have been extensively investigatedbecause of their biocompatibility and biodegradability. Nanoparticlesand other nanocarriers act as potential carries for several classes ofdrugs such as anticancer agents, antihypertensive agents,immunomodulators, and hormones; and macromolecules such as nucleicacids, proteins, peptides, and antibodies. See, e.g., Crit. Rev. Ther.Drug Carrier Syst. 21:387-422, 2004; Nanomedicine: Nanotechnology,Biology and Medicine 1:22-30, 2005.

Pharmaceutical Compositions

The term “pharmaceutical” as used herein refers to a chemical substanceintended for use in the cure, treatment, or prevention of disease andwhich is subject to an approval process by the U.S. Food and DrugAdministration (or a non-U.S. equivalent thereof) as a prescription orover-the-counter drug product. Details on techniques for formulation andadministration of such compositions may be found in Remington, TheScience and Practice of Pharmacy 21^(st) Edition (Mack Publishing Co.,Easton, Pa.) and Nielloud and Marti-Mestres, Pharmaceutical Emulsionsand Suspensions: 2^(nd) Edition (Marcel Dekker, Inc, New York).

For the purposes of this disclosure, the pharmaceutical compositions maybe administered by a variety of means including orally, parenterally, byinhalation spray, topically, or rectally in formulations containingpharmaceutically acceptable carriers, adjuvants and vehicles. The termparenteral as used here includes but is not limited to subcutaneous,intravenous, intramuscular, intraarterial, intradermal, intrathecal andepidural injections with a variety of infusion techniques. Intraarterialand intravenous injection as used herein includes administration throughcatheters. Administration via intracoronary stents and intracoronaryreservoirs is also contemplated. The term oral as used herein includes,but is not limited to oral ingestion, or delivery by a sublingual orbuccal route. Oral administration includes fluid drinks, energy bars, aswell as pill formulations.

Pharmaceutical compositions may be in any form suitable for the intendedmethod of administration. When used for oral use for example, tablets,troches, lozenges, aqueous or oil suspensions, dispersible powders orgranules, emulsions, hard or soft capsules, syrups or elixirs may beprepared. Compositions intended for oral use may be prepared accordingto any method known to the art for the manufacture of pharmaceuticalcompositions and such compositions may contain one or more agentsincluding sweetening agents, flavoring agents, coloring agents andpreserving agents, in order to provide a palatable preparation. Tabletscontaining a drug compound in admixture with non-toxic pharmaceuticallyacceptable excipient which are suitable for manufacture of tablets areacceptable. These excipients may be, for example, inert diluents, suchas calcium or sodium carbonate, lactose, calcium or sodium phosphate;granulating and disintegrating agents, such as maize starch, or alginicacid; binding agents, such as starch, gelatin or acacia; and lubricatingagents; such as magnesium stearate, stearic acid or talc. Tablets may beuncoated, or may be coated by known techniques including entericcoating, colonic coating, or microencapsulation to delay disintegrationand adsorption in the gastrointestinal tract and/or provide a sustainedaction over a longer period. For example, a time delay material such asglyceryl monostearate or glyceryl distearate alone or with a wax may beemployed.

Formulations for oral use may be also presented as hard gelatin capsuleswhere the drug compound is mixed with an inert solid diluent, forexample calcium phosphate or kaolin, or as soft gelatin capsules whereinthe active ingredient is mixed with water or an oil medium, such aspeanut oil, liquid paraffin or olive oil.

Pharmaceutical compositions may be formulated as aqueous suspensions inadmixture with excipients suitable for the manufacture ofaqueous-suspensions. Such excipients include a suspending agent, such assodium carboxymethylcellulose, methylcellulose, hydroxypropylmethylcellulose, sodium alginate, polyvinylpyrrolidone, gum tragacanthand gum acacia, and dispersing or wetting agents such as a naturallyoccurring phosphatide (e.g., lecithin), a condensation product of analkylene oxide with a fatty acid (e.g., polyoxyethylene stearate), acondensation product of ethylene oxide with a long chain aliphaticalcohol (e.g., heptadecaethyleneoxycetanol), a condensation product ofethylene oxide with a partial ester derived from a fatty acid and ahexitol anhydride (e.g., polyoxyethylene sorbitan monooleate). Theaqueous suspension may also contain one or more preservatives such asethyl or n-propyl p-hydroxy-benzoate, one or more coloring agents, oneor more flavoring agents and one or more sweetening agents, such assucrose or saccharin.

Oil suspensions may be formulated by suspending the active ingredient ina vegetable oil, such as arachis oil, olive oil, sesame oil or coconutoil, or a mineral oil such as liquid paraffin. The oral suspensions maycontain a thickening agent, such as beeswax, hard paraffin or cetylalcohol. Sweetening agents, such as those set forth above, and flavoringagents may be added to provide a palatable oral preparation. Thesecompositions may be preserved by the addition of an antioxidant such asascorbic acid.

Dispersible powders and granules of the disclosure suitable forpreparation of an aqueous suspension by the addition of water providethe active ingredient in admixture with a dispersing or wetting agent, asuspending agent, and one or more preservatives. Suitable dispersing orwetting agents and suspending agents are exemplified by those disclosedabove. Additional excipients, for example sweetening, flavoring andcoloring agents, may also be present.

The pharmaceutical compositions of the disclosure may also be in theform of oil-in-water emulsions. The oily phase may be a vegetable oil,such as olive oil or arachis oil, a mineral oil, such as liquidparaffin, or a mixture of these. Suitable emulsifying agents includenaturally-occurring gums, such as gum acacia and gum tragacanth,naturally occurring phosphatides, such as soybean lecithin, esters orpartial esters derived from fatty acids and hexitol anhydrides, such assorbitan monooleate, and condensation products of these partial esterswith ethylene oxide, such as polyoxyethylene sorbitan monooleate. Theemulsion may also contain sweetening and flavoring agents.

Syrups and elixirs may be formulated with sweetening agents, such asglycerol, sorbitol or sucrose. Such formulations may also contain ademulcent, a preservative, a flavoring or a coloring agent.

The pharmaceutical compositions of the disclosure may be in the form ofa sterile injectable preparation, such as a sterile injectable aqueousor oleaginous suspension. This suspension may be formulated according tothe known art using those suitable dispersing or wetting agents andsuspending agents which have been mentioned above. The sterileinjectable preparation may also be a sterile injectable solution orsuspension in a non-toxic parenterally acceptable diluent or solventsuch as a solution in 1,3-butane-diol or prepared as a lyophilizedpowder. Among the acceptable vehicles and solvents that may be employedare water, Ringer's solution and isotonic sodium chloride solution. Inaddition, sterile fixed oils may conventionally be employed as a solventor suspending medium. For this purpose any bland fixed oil may beemployed including synthetic mono- or diglycerides. In addition, fattyacids such as oleic acid may likewise be used in the preparation ofinjectables.

The amount of active ingredient that may be combined with the carriermaterial to produce a single dosage form will vary depending upon thehost treated and the particular mode of administration. For example, atime-release formulation intended for oral administration to humans maycontain approximately 20 to 500 mg of active material compounded with anappropriate and convenient amount of carrier material which may varyfrom about 5 to about 95% of the total compositions. It is preferredthat the pharmaceutical composition be prepared which provides easilymeasurable amounts for administration. Typically, an effective amount tobe administered systemically is about 0.1 mg/kg to about 100 mg/kg anddepends upon a number of factors including, for example, the age andweight of the subject (e.g., a mammal such as a human), the precisecondition requiring treatment and its severity, the route ofadministration, and will ultimately be at the discretion of theattendant physician or veterinarian. It will be understood, however,that the specific dose level for any particular patient will depend on avariety of factors including the activity of the specific compoundemployed, the age, body weight, general health, sex and diet of theindividual being treated; the time and route of administration; the rateof excretion; other drugs which have previously been administered; andthe severity of the particular condition undergoing therapy, as is wellunderstood by those skilled in the art.

As noted above, formulations of the disclosure suitable for oraladministration may be presented as discrete units such as capsules,cachets or tablets each containing a predetermined amount of the activeingredient, as a powder or granules; as a solution or a suspension in anaqueous or non-aqueous liquid, or as an oil-in-water liquid emulsion ora water-in-oil liquid emulsion. The pharmaceutical compositions may alsobe administered as a bolus, electuary or paste.

A tablet may be made by compression or molding, optionally with one ormore accessory ingredients. Compressed tablets may be prepared bycompressing in a suitable machine the active ingredient in a freeflowing form such as a powder or granules, optionally mixed with abinder (e.g., povidone, gelatin, hydroxypropyl ethyl cellulose),lubricant, inert diluent, preservative, disintegrant (e.g., sodiumstarch glycolate, cross-linked povidone, cross-linked sodiumcarboxymethyl cellulose) surface active or dispersing agent. Moldedtablets may be made in a suitable machine using a mixture of thepowdered compound moistened with an inert liquid diluent. The tabletsmay optionally be coated or scored and may be formulated so as toprovide slow or controlled release of the active ingredient thereinusing, for example, hydroxypropyl methylcellulose in varying proportionsto provide the desired release profile. Tablets may optionally beprovided with an enteric or colonic coating to provide release in partsof the gut other than the stomach. This is particularly advantageouswith the compounds of formula 1 when such compounds are susceptible toacid hydrolysis.

Formulations suitable for topical administration in the mouth includelozenges comprising the active ingredient in a flavored base, usuallysucrose and acacia or tragacanth; pastilles comprising the activeingredient in an inert base such as gelatin and glycerin, or sucrose andacacia; and mouthwashes comprising the active ingredient in a suitableliquid carrier.

Formulations for rectal administration may be presented as a suppositorywith a suitable base comprising for example cocoa butter or asalicylate.

Formulations suitable for vaginal administration may be presented aspessaries, tampons, creams, gels, pastes, foams or spray formulationscontaining in addition to the active ingredient such carriers as areknown in the art to be appropriate.

Formulations suitable for parenteral administration include aqueous andnon-aqueous isotonic sterile injection solutions which may containantioxidants, buffers, bacteriostats and solutes which render theformulation isotonic with the blood of the intended recipient; andaqueous and non-aqueous sterile suspensions which may include suspendingagents and thickening agents. The formulations may be presented inunit-dose or multi-dose sealed containers, for example, ampoules andvials, and may be stored in a freeze-dried (lyophilized) conditionrequiring only the addition of the sterile liquid carrier, for examplewater for injections, immediately prior to use. Injection solutions andsuspensions may be prepared from sterile powders, granules and tabletsof the kind previously described.

As used herein, pharmaceutically acceptable salts include, but are notlimited to: acetate, pyridine, ammonium, piperazine, diethylamine,nicotinamide, formic, urea, sodium, potassium, calcium, magnesium, zinc,lithium, cinnamic, methylamino, methanesulfonic, picric, tartaric,triethylamino, dimethylamino, and tris(hydoxymethyl)aminomethane.Additional pharmaceutically acceptable salts are known to those skilledin the art.

An effective amount for a particular patient may vary depending onfactors such as the condition being treated, the overall health of thepatient, the route and dose of administration and the severity of sideeffects. Guidance for methods of treatment and diagnosis is available(see, e.g., Maynard, et al. (1996) A Handbook of SOPs for Good ClinicalPractice, Interpharm Press, Boca Raton, Fla.; Dent (2001) GoodLaboratory and Good Clinical Practice, Urch Publ., London, UK).

An effective amount may be given in one dose, but is not restricted toone dose. Thus, the administration can be two, three, four, five, six,seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen,sixteen, seventeen, eighteen, nineteen, twenty, or more, administrationsof pharmaceutical composition. Where there is more than oneadministration of a pharmaceutical composition in the present methods,the administrations can be spaced by time intervals of one minute, twominutes, three, four, five, six, seven, eight, nine, ten, or moreminutes, by intervals of about one hour, two hours, three, four, five,six, seven, eight, nine, ten, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,21, 22, 23, 24 hours, and so on. In the context of hours, the term“about” means plus or minus any time interval within 30 minutes. Theadministrations can also be spaced by time intervals of one day, twodays, three days, four days, five days, six days, seven days, eightdays, nine days, ten days, 11 days, 12 days, 13 days, 14 days, 15 days,16 days, 17 days, 18 days, 19 days, 20 days, 21 days, and combinationsthereof. The invention is not limited to dosing intervals that arespaced equally in time, but encompass doses at non-equal intervals.

A dosing schedule of, for example, once/week, twice/week, threetimes/week, four times/week, five times/week, six times/week, seventimes/week, once every two weeks, once every three weeks, once everyfour weeks, once every five weeks, and the like, is available for theinvention. The dosing schedules encompass dosing for a total period oftime of, for example, one week, two weeks, three weeks, four weeks, fiveweeks, six weeks, two months, three months, four months, five months,six months, seven months, eight months, nine months, ten months, elevenmonths, and twelve months.

Provided are cycles of the above dosing schedules. The cycle can berepeated about, e.g., every seven days; every 14 days; every 21 days;every 28 days; every 35 days; 42 days; every 49 days; every 56 days;every 63 days; every 70 days; and the like. An interval of non dosingcan occur between a cycle, where the interval can be about, e.g., sevendays; 14 days; 21 days; 28 days; 35 days; 42 days; 49 days; 56 days; 63days; 70 days; and the like. In this context, the term “about” meansplus or minus one day, plus or minus two days, plus or minus three days,plus or minus four days, plus or minus five days, plus or minus sixdays, or plus or minus seven days.

Methods for co-administration with an additional therapeutic agent arewell known in the art (Hardman, et al. (eds.) (2001) Goodman andGilman's The Pharmacological Basis of Therapeutics, 10th ed.,McGraw-Hill, New York, N.Y.; Poole and Peterson (eds.) (2001)Pharmacotherapeutics for Advanced Practice: A Practical Approach,Lippincott, Williams & Wilkins, Phila., Pa.; Chabner and Longo (eds.)(2001) Cancer Chemotherapy and Biotherapy, Lippincott, Williams &Wilkins, Phila., Pa.).

As noted, the compositions of the present invention are preferablyformulated as pharmaceutical compositions for parenteral or enteraldelivery. A typical pharmaceutical composition for administration to ananimal comprises a pharmaceutically acceptable vehicle such as aqueoussolutions, non-toxic excipients, including salts, preservatives, buffersand the like. See, e.g., Remington's Pharmaceutical Sciences, 15th Ed.,Easton ed., Mack Publishing Co., pp 1405-1412 and 1461-1487 (1975); TheNational Formulary XIV, 14th Ed., American Pharmaceutical Association,Washington, D.C. (1975). Examples of non-aqueous solvents are propyleneglycol, polyethylene glycol, vegetable oil and injectable organic esterssuch as ethyloleate. Aqueous carriers include water, alcoholic/aqueoussolutions, saline solutions, parenteral vehicles such as sodiumchloride, Ringer's dextrose, etc. Intravenous vehicles include fluid andnutrient replenishers. Preservatives include antimicrobial agents,anti-oxidants, chelating agents and inert gases. The pH and exactconcentration of the various components the pharmaceutical compositionare adjusted according to routine skills in the art.

Examples

The following examples serve to illustrate the present invention. Theseexamples are in no way intended to limit the scope of the invention.

Example 1 General Methods

Anhydrous solvents and reagents suitable for solution phaseoligonucleotide synthesis were purchased and handled under dry argon ornitrogen using anhydrous technique. Amidite coupling reactions andcyclizations were carried out in anhydrous acetonitrile or pyridineunder dry argon or nitrogen. The starting materials for all reactions indry pyridine were dried by concentration (three times) from pyridine.Preparative silica gel flash chromatography was carried out using Fluka60A high-purity grade or Merck Grade 9385 silica using gradients ofmethanol in dichloromethane. Analytical HPLC was carried out on a VarianProStar 210 HPLC system with a ProStar 330 photodiode array detectormonitoring at 254 nm using a either a Varian Microsorb 10 micron C18250×4.6 mm or a Varian 3micronC18 100×4.6 mm column and gradients of 10mM TEAA and acetonitrile. Preparative HPLC was carried out on a Shimadzupreparative LC20-AP HPLC system, equipped with a SPD-20A UV/Vis detectormonitoring at 254 nm on a Varian Microsorb 60-8 C-18 41.6×250 mm columnusing gradients of 10 mM TEAA and acetonitrile at a flow rate of 50ml/min Solid phase extractions using C-18 Sep-Pak (Waters) were carriedout at loadings of 3% (wt/wt). LC/MS (ESI/APCI) was obtained on a singlequadrapole Shimadzu 2010EV instrument with PDA, MS, and ELSD detectionusing a Shimadzu LC20D analytical HPLC. High resolution FT-ICR mass specwas obtained from both WM Keck Foundation Biotechnology ResourceLaboratory at Yale University in New Haven, Conn., and the QB3/ChemistryMass Spect Lab at UC Berkeley.

¹H, ³¹P, ¹H-¹H COSY (2D NMR correlation spectroscopy), ¹H-³¹P HMBC(heteronuclear multiple-bond correlation spectroscopy) spectra wereacquired in d6-DMSO with 10 uL D₂O (16 hr delay after D₂O addition) at45° C. on a Varian INOVA-500 NMR spectrometer operating at 500 MHz for1H and 202 MHz for 31P. The resulting FIDs were transferred to a PC andprocessed using NUTS NMR processing software from Acorn NMR Inc. Thechemical shifts were referenced to the DMSO solvent, 2.50 ppm for 1H.Per IUPAC recommendations for referencing of NMR spectral, the 31Pchemical shifts were referenced using the “unified scale” to theabsolute 1H frequency of 0 ppm. Some of the 1H and 31P spectra wereacquired on a JEOL ECX-400 NMR spectrometer operating at 400 MHz for 1Hand 162 MHz for 31P. The gradient COSY spectra were acquired at 45.0° C.on a Varian INOVA-500 NMR spectrometer operating at 500 MHz for 1H and202 MHz for 31P. The resulting FIDs were transferred to a PC andprocessed using NUTS NMR processing software from Acorn NMR Inc. Thechemical shifts were referenced to the DMSO solvent, 2.50 ppm for 1H.Per IUPAC recommendations for referencing of NMR spectral, the 31Pchemical shifts were referenced using the “unified scale” to theabsolute 1H frequency of 0 ppm. The gradient COSY spectrum was acquiredin absolute value mode using 2048 data points in the direct dimensionand 256 time points in the indirect dimension. Both dimensions wereapodized using sinebell squared functions. The indirect dimension waszero filled to give a final matrix size of 2048×2048 points and aresolution of 3.91 Hz/data point in both dimensions.

Assignment of regiochemistry at phosphodiester linkage: 1H-1H COSY incombination with ¹H-³¹P HMBC experiments were used to provide directevidence that the regiochemistry of the phosphodiester linkages are 2′,5′-3′, 5′ (see for example FIGS. 3C and 3D).

Abbreviations and Acronyms. Guanine=G. isobutyryl guanine=G^(ib).4,4-dimethoxytrityl=DMT. OCH₂CH₂CH₃=CEO. tert-butyldimethylsilyl=TBS.adenine=A. benzoyl adenine=A^(Bz)′,2′-O-myristoyl-cyclic-[G(2′,5′)pG(3′,5′)p]=C14-ML-CDG=10 (TEA salt). AllCDN products were ≧95% pure as indicated by C18 reverse phase HPLCanalysis using UV detection at 254 nm (see FIG. 2E for purity ofstructure 8).

Example 2 Synthesis of 2′-O-propargyl-cyclic-A(2′,5′)pA(3′,5′)p(2′-O-propargyl-ML-CDA, structure 8), FIG. 2).

1) Preparation of 3.

To a solution of 1.7 g (1.72 mmol)N⁶-benzoyl-5′-O-(4,4′-dimethoxytrityl)-3′-O-tert-butyldimethylsilyl-2′-O-[(2-cyanoethyl)-N,N-diisopropylaminophinyl]adenosine(1) in 7.5 ml acetonitrile was added 0.054 ml (3 mmole) water and 0.35 g(1.8 mmole) pyridinium trifluoroacetate. After 5 minutes stirring atroom temperature 7.5 ml tert-butylamine was added and the reactionstirred for 15 minutes at room temperature. The solvents were removedunder reduced pressure to give 2 as a foam which was then co-evaporatedwith acetonitrile (3×15 ml), then dissolved in 18 ml dichloromethane. Tothis solution was added water (0.27 ml, 15 mmole) and 18 ml of 6% (v/v)dichloroacetic acid (13.2 mmole) in dichloromethane. After 10 minutes atroom temperature the reaction was quenched by the addition of pyridine(2.1 ml, 26 mmole), and concentrated to an oil which was dried by threeco-evaporations with 12 ml anhydrous acetonitrile, the last time leaving3 in a volume of ˜4 ml.

2) Preparation of a Dry Solution of 4.

N⁶-benzoyl-5′-O-(4,4′-dimethoxytrityl)-2′-O-propargyl-3′-O-[(2-cyanoethyl)-N,N-diisopropylaminophinyl]adenosine(4, 2 g, 2.2 mmole) was dissolved in 25 ml anhydrous acetonitrile anddried by three co-evaporations with 25 ml anhydrous acetonitrile, thelast time leaving ˜6 ml. Ten 3 Å molecular sieves were added and thedried solution stored under argon until use.

3) Preparation of 2′,5′-Linear Dimer 5.

Azeo dried 4 (2 g, 2.2 mmole) in ˜6 ml acetonitrile was added viasyringe to a solution of 3 (1.72 mmole) in ˜4 ml of anhydrousacetonitrile. After 5 minutes stirring at room temperature, 0.82 ml (4.5mmole) of 5.5M tert-butyl hydroperoxide in decane was added and thereaction stirred for 30 minutes at room temperature. The reaction wascooled in an ice bath, and 0.38 g NaHSO₃ in 0.75 ml water was added andstirred at room temperature for 5 minutes. The reaction was concentratedand the residual oil dissolved in 24 ml dichloromethane. Water (0.27 ml,15 mmole) and 24 ml of 6% (v/v) dichloroacetic acid (17.4 mmole) indichloromethane was added, and the reaction stirred for 10 minutes atroom temperature. 15 ml pyridine was added to quench the dichloroaceticacid, which was then concentrated down to ˜4 ml.

4) Cyclization and Oxidation of 5 to Give theFully-Protected-Propargyl-Cyclic-Dinucleotide 6.

5 was dissolved in 45 ml dry pyridine which was concentrated down to avolume of approximately 30 ml.2-chloro-5,5-dimethyl-1,3,2-dioxaphosphorinane-2-oxide (DMOCP, 1 g, 5.2mmole) was then added and the reaction stirred for 10 minutes at roomtemperature. 1 ml water was added immediately followed by addition of I₂(0.5 g, 2 mmole), and the reaction stirred for 5 minutes at roomtemperature. The reaction mix was then poured into 210 ml watercontaining 0.3 g NaHSO₃ and stirred for 5 minutes at room temperature. 6g NaHCO₃ was slowly added and stirred for 5 minutes at room temperature,then poured into a separatory funnel and extracted with 250 ml 1:1 ethylacetate:diethyl ether. The aqueous layer was extracted again with 60 ml1:1 ethyl acetate:diethyl ether. The organic layers were combined andconcentrated under reduced pressure to yield approximately 5.6 g of anoil containing fully-protected-propargyl cyclic dinucleotide 6.

6) Deprotection of the Fully-Protected-Propargyl Cyclic Dinucleotide 6to Crude 7.

5.6 g of crude 6 was transferred to a thick-walled glass pressure tube.30 ml methanol and 30 ml concentrated aqueous ammonia was added and thetube was heated with stirring in an oil bath at 55° C. for 4 h, at whichtime analytical HPLC showed deprotection was complete. The reactionmixture was cooled to near ambient temperature, sparged with a stream ofargon gas for 30 minutes, and then transferred to a large round bottomflask. Most of the volatiles were removed under reduced pressure to givea residue of 4.7 g, which was triturated against 20 ml 1:1 (v/v)dichloromethane:hexane. Any remaining solvent was removed under reducedpressure to give 4.5 g of a solid containing 7.

7) Preparative HPLC Purification of Crude 7 to Give Pure 7.

The crude solid containing 7 was taken up in 25 ml of CH₃CN/water (1:1).After 0.45 micron PTFE filtration, 4-5 ml sample portions were appliedto a C-18 Dynamax column (40×250 mm) Elution was performed with agradient of acetonitrile and 10 mM aqueous triethylammonium acetate (20%to 50% CH₃CN over 20 minutes at 50 ml/min flow). Fractions from thepreparative HPLC runs containing pure 7 were pooled, evaporated toremove most of the CH₃CN and water and coevaporated several times withCH₃CN to give 55 mg of pure 7.

8) Deprotection of the TBS Group of 7 with TriethylamineTrihydrofluoride, Neutralization with TEAB, Solid Phase Extraction witha C-18 Sep-Pak and Lyophilization to Give Pure 8 as theBis-Triethylammonium Salt.

To 55 mg of 7 was added 1.0 ml of neat triethylamine trihydrofluoride.The mixture was stirred at room temperature for approximately 3 h. Themixture was then transferred to an oil bath at 50° C. for an additional2 hours, at which time analytical HPLC confirmed completion of thereaction. The sample was neutralized by dropwise addition into 5 ml ofchilled, stirred 1M triethylammonium bicarbonate. Approximately 1-2 mlTEA was added dropwise to the stirred, chilled solution until pH papershowed neutral/slightly basic (˜pH8) conditions were achieved. Theneutralized solution was desalted on a Waters C-18 Sep-Pak and theproduct eluted with CH₃CN/10 mM aqueous triethylammonium acetate(15:85). The CH₃CN was evaporated under reduced pressure and thesolution was frozen and lyophilized. An additional round oflyophilization from water gave 9 mg (13 μmole) of 2′-O-propargyl-ML-CDA(8) as the bis-triethylammonium salt. ¹H NMR (500 MHz, 45° C.,DMSO-D₆+15 μL D₂O) δ 8.68 (s, 1H), 8.31 (s, 1H), 8.15 (s, 1H), 8.14 (s,1H), 6.10 (d, J=8.0, 1H), 5.99 (d, J=6.0, 1H), 5.06-5.04 (m, 1H),4.98-4.94 (m, 1H), 4.53 (qt, J=16.0, 2.5, 2H), 4.39 (d, J=4.0, 1H),4.27-4.26 (m, 1H), 4.14-4.13 (m, 1H), 4.05-3.90 (m, 3H), 3.74 (d,J=12.0, 1H), 3.21 (t, J=2.5, 1H), 3.03 (q, J=7.0, 12H), 1.14 (t, J=7.5,19H); ³¹P NMR (200 MHz, 45° C., DMSO-D₆+15 μL D₂O) δ −1.48, −1.82 (FIG.3A-3D); HRMS (FT-ICR) m/z: [M-H]⁻ calcd for C₂₃H₂₅N₁₀O₁₂P₂ 695.1134.found 695.1118.

FIGS. 4-6 depict alternative compounds which may be made by analogousmethods to those described herein.

Example 3 Inhibition of STING-Dependent Responses

To evaluate if the antagonist 2′-O-propargyl-cyclic-[A(2′,5′)pA(3′,5′)p](ML-propargyl-CDA) can inhibit STING-dependent induction of type Iinterferon induction by Rp, Rp dithio cyclic [A(2′,5′)pA(3′,5′)p] (MLRR-CDA) in human cells, 4×10⁵ THP1-Blue™ ISG cells (a human monocytecell line transfected with an IRF-inducible secreted embryonic alkalinephosphatase reporter gene (Invivogen) which express alkaline phosphataseunder the control of a promoter comprised of five IFN-stimulatedresponse elements) were incubated with 50 μM of Rp, Rp dithio cyclic[A(2′,5′)pA(3′,5′)p] (ML RR-CDA), 10 μM or 50 μM of the antagonist2′-O-propargyl-cyclic-[A(2′,5′)pA(3′,5′)p] (ML-propargyl-CDA), both 50μM Rp, Rp dithio cyclic [A(2′,5′)pA(3′,5′)p] (ML RR-CDA) and 10 μM or 50μM 2′-O-propargyl-cyclic-[A(2′,5′)pA(3′,5′)p] (ML-propargyl-CDA), or 50μM Rp, Rp dithio cyclic [A(2′,5′)pA(3′,5′)p] (ML RR-CDA) after a 30 minpre-incubation with 10 μM 2′-O-propargyl-cyclic-[A(2′,5′)pA(3′,5′)p](ML-propargyl-CDA). After 30 minutes, cells were washed and plated in96-well dish in RPMI media containing 10% FBS, and incubated at 37° C.with 5% CO₂. Cell culture supernatants from each sample were collectedafter 16 hr incubation, and 20 μL of the cell culture supernatants wasadded to 180 μL QUANTI-Blue reagent (Invivogen) and incubated for 5minutes to evaluate type I interferon protein levels. Readings atAbsorbance 655 nm were measured with a Versa Max kineticspectrophotometer (Molecular Diagnostics).

As shown in FIG. 7A, addition of 10 μM or 50 μM of the antagonist2′-O-propargyl-cyclic-[A(2′,5′)pA(3′,5′)p] (ML-propargyl-CDA) with 50 μMRp, Rp dithio cyclic [A(2′,5′)pA(3′,5′)p] (ML RR-CDA) significantlyinhibited the induction of type I IFN by Rp, Rp dithio cyclic[A(2′,5′)pA(3′,5′)p] (ML RR-CDA) in a dose-dependent manner. FIG. 7Bshows that pre-incubation with 10 μM2′-O-propargyl-cyclic-[A(2′,5′)pA(3′,5′)p] (ML-propargyl-CDA) inhibitsinduction of type I interferon by the subsequent addition of 50 μM Rp,Rp dithio cyclic [A(2′,5′)pA(3′,5′)p] (ML RR-CDA). It is known thatcyclic dinucleotides such as Rp, Rp dithio cyclic [A(2′,5′)pA(3′,5′)p](ML RR-CDA) induce type I IFN signaling via STING. Therefore, thereduction in Rp, Rp dithio cyclic [A(2′,5′)pA(3′,5′)p] (MLRR-CDA)-induced type I IFN production by2′-O-propargyl-cyclic-[A(2′,5′)pA(3′,5′)p] (ML-propargyl-CDA)demonstrates that 2′-O-propargyl-cyclic-[A(2′,5′)pA(3′,5′)p](ML-propargyl-CDA) is an antagonist of human STING.

Structural studies of apo and cyclic di-nucleotide-bound forms of STINGhave shown that STING forms a symmetrical v-shaped dimer in the free andbound states with the cyclic di-nucleotide bound in a pocket formed bythe dimer interface (Gao P., et al., (2013). Cell 154, 748-762 andreviewed in Burdette and Vance, (2013) Nature Immunology 14, 19-26).STING undergoes a large conformational switch upon ligand binding from amore “open” conformation in the apo form to a “closed” conformation witha four-stranded antiparallel β sheet lid forming over theligand-binding.

The results shown here demonstrate that2′-O-propargyl-cyclic-[A(2′,5′)pA(3′,5′)p] (ML-propargyl-CDA) can bindin the same binding pocket formed by the interface of two STING dimers.Binding of 2′-O-propargyl-cyclic-[A(2′,5′)pA(3′,5′)p] (ML-propargyl-CDA)within the pocket would prevent the binding of an activating cyclicdi-nucleotide. The propargyl group on2′-O-propargyl-cyclic-[A(2′,5′)pA(3′,5′)p] (ML-propargyl-CDA) extendingfrom the antagonist molecule residing in the STING binding pocketsterically blocks formation of the antiparallel β sheet lid over theligand-binding pocket, thereby preventing STING from transitioning tothe signaling competent conformation. Collectively these resultsindicate that 2′-O-propargyl-cyclic-[A(2′,5′)pA(3′,5′)p](ML-propargyl-CDA) is capable of inhibiting the activity of human STING.

One skilled in the art readily appreciates that the present invention iswell adapted to carry out the objects and obtain the ends and advantagesmentioned, as well as those inherent therein. The examples providedherein are representative of preferred embodiments, are exemplary, andare not intended as limitations on the scope of the invention.

It is to be understood that the invention is not limited in itsapplication to the details of construction and to the arrangements ofthe components set forth in the following description or illustrated inthe drawings. The invention is capable of embodiments in addition tothose described and of being practiced and carried out in various ways.Also, it is to be understood that the phraseology and terminologyemployed herein, as well as the abstract, are for the purpose ofdescription and should not be regarded as limiting.

As such, those skilled in the art will appreciate that the conceptionupon which this disclosure is based may readily be utilized as a basisfor the designing of other structures, methods and systems for carryingout the several purposes of the present invention. It is important,therefore, that the claims be regarded as including such equivalentconstructions insofar as they do not depart from the spirit and scope ofthe present invention.

While the invention has been described and exemplified in sufficientdetail for those skilled in this art to make and use it, variousalternatives, modifications, and improvements should be apparent withoutdeparting from the spirit and scope of the invention. The examplesprovided herein are representative of preferred embodiments, areexemplary, and are not intended as limitations on the scope of theinvention. Modifications therein and other uses will occur to thoseskilled in the art. These modifications are encompassed within thespirit of the invention and are defined by the scope of the claims.

It will be readily apparent to a person skilled in the art that varyingsubstitutions and modifications may be made to the invention disclosedherein without departing from the scope and spirit of the invention.

All patents and publications mentioned in the specification areindicative of the levels of those of ordinary skill in the art to whichthe invention pertains. All patents and publications are hereinincorporated by reference to the same extent as if each individualpublication was specifically and individually indicated to beincorporated by reference.

The invention illustratively described herein suitably may be practicedin the absence of any element or elements, limitation or limitationswhich is not specifically disclosed herein. Thus, for example, in eachinstance herein any of the terms “comprising”, “consisting essentiallyof” and “consisting of” may be replaced with either of the other twoterms. The terms and expressions which have been employed are used asterms of description and not of limitation, and there is no intentionthat in the use of such terms and expressions of excluding anyequivalents of the features shown and described or portions thereof, butit is recognized that various modifications are possible within thescope of the invention claimed. Thus, it should be understood thatalthough the present invention has been specifically disclosed bypreferred embodiments and optional features, modification and variationof the concepts herein disclosed may be resorted to by those skilled inthe art, and that such modifications and variations are considered to bewithin the scope of this invention as defined by the appended claims.

Other embodiments are set forth within the following claims.

1. A composition comprising: one or more cyclic purine dinucleotidesthat inhibit STING-dependent type I Interferon production.
 2. Acomposition according to claim 1, wherein the cyclic purinedinucleotides present in the composition have the structure:

covalently linked to

wherein R3 is a covalent bond to the 5′ carbon of (b), R4 is a covalentbond to the 2′ or 3′ carbon of (b), R1 is a purine linked through its N9nitrogen to the ribose ring of (a), R5 is a purine linked through its N9nitrogen to the ribose ring of (b), Each of X₁ and X₂ are independentlyO or S, R2 is H or an optionally substituted straight chain alkyl offrom 1 to 18 carbons and from 0 to 3 heteroatoms, an optionallysubstituted alkenyl of from 1-9 carbons, an optionally substitutedalkynyl of from 1-9 carbons, or an optionally substituted aryl, whereinsubstitution(s), when present, may be independently selected from thegroup consisting of C₁₋₆ alkyl straight or branched chain, benzyl,halogen, trihalomethyl, C₁₋₆ alkoxy, —NO₂, —NH₂, —OH, ═O, —COOR′ whereR′ is H or lower alkyl, —CH₂OH, and —CONH₂, the 2′ or 3′ carbon of (b)which is not in a covalent bond with (a) is —O—R6, wherein R6 is H or anoptionally substituted straight chain alkyl of from 1 to 18 carbons andfrom 0 to 3 heteroatoms, an optionally substituted alkenyl of from 1-9carbons, an optionally substituted alkynyl of from 1-9 carbons, or anoptionally substituted aryl, wherein substitution(s), when present, maybe independently selected from the group consisting of C₁₋₆ alkylstraight or branched chain, benzyl, halogen, trihalomethyl, C₁₋₆ alkoxy,—NO₂, —NH₂, —OH, ═O, —COOR′ where R′ is H or lower alkyl, —CH₂OH, and—CONH₂, and wherein R2 and R6 are not both H, or prodrugs orpharmaceutically acceptable salts thereof.
 3. A composition according toclaim 1, wherein the cyclic purine dinucleotides present in thecomposition have the structure:

covalently linked to

wherein R3 is a covalent bond to the 5′ carbon of (b), R4 is a covalentbond to the 3′ carbon of (b), R1 is a purine linked through its N9nitrogen to the ribose ring of (a), R5 is a purine linked through its N9nitrogen to the ribose ring of (b), Each of X₁ and X₂ are independently0 or S, R2 is H or an optionally substituted straight chain alkyl offrom 1 to 18 carbons and from 0 to 3 heteroatoms, an optionallysubstituted alkenyl of from 1-9 carbons, an optionally substitutedalkynyl of from 1-9 carbons, or an optionally substituted aryl, whereinsubstitution(s), when present, may be independently selected from thegroup consisting of C₁₋₆ alkyl straight or branched chain, benzyl,halogen, trihalomethyl, C₁₋₆ alkoxy, —NO₂, —NH₂, —OH, ═O, —COOR′ whereR′ is H or lower alkyl, —CH₂OH, and —CONH₂, the 2′ carbon of (b) is—O—R6, wherein R6 is H or an optionally substituted straight chain alkylof from 1 to 18 carbons and from 0 to 3 heteroatoms, an optionallysubstituted alkenyl of from 1-9 carbons, an optionally substitutedalkynyl of from 1-9 carbons, or an optionally substituted aryl, whereinsubstitution(s), when present, may be independently selected from thegroup consisting of C₁₋₆ alkyl straight or branched chain, benzyl,halogen, trihalomethyl, C₁₋₆ alkoxy, —NO₂, —NH₂, —OH, ═O, —COOR′ whereR′ is H or lower alkyl, —CH₂OH, and —CONH₂, and wherein R2 and R6 arenot both H, or prodrugs or pharmaceutically acceptable salts thereof. 4.A substantially pure cyclic purine dinucleotide composition according toclaim 1, wherein one or both of R2 and R6 are independently anunsubstituted straight chain alkyl of from 1 to 18 carbons, anunsubstituted alkenyl of from 1-9 carbons, an unsubstituted alkynyl offrom 1-9 carbons, or an unsubstituted aryl, and most preferably selectedfrom the group consisting of selected from the group consisting ofallyl, propargyl, homoallyl, homopropargyl, methyl, ethyl, propyl,isopropyl, isobutyl, cyclopropylmethyl, and benzyl.
 5. A substantiallypure cyclic purine dinucleotide composition according to claim 1,wherein one or both of R2 and R6 are allyl.
 6. A substantially purecyclic purine dinucleotide composition according to claim 1, wherein oneor both of R2 and R6 comprise a propargyl.
 7. A substantially purecyclic purine dinucleotide composition according to claim 1, wherein oneor both of R2 and R6 are methyl.
 8. A substantially pure cyclic purinedinucleotide composition according to claim 1, wherein one or both of R2and R6 are ethyl.
 9. A substantially pure cyclic purine dinucleotidecomposition according to claim 1, wherein one or both of R2 and R6 arepropyl.
 10. A substantially pure cyclic purine dinucleotide compositionaccording to claim 1, wherein one or both of R2 and R6 are benzyl.
 11. Asubstantially pure cyclic purine dinucleotide composition according toclaim 1, wherein one of R2 or R6 is selected from the group consistingof allyl, propargyl, homoallyl, homopropargyl, methyl, ethyl, propyl,isopropyl, isobutyl, cyclopropylmethyl, and benzyl, and the other of R2or R6 comprises a prodrug leaving group.
 12. A substantially pure cyclicpurine dinucleotide composition according to claim 11, wherein theprodrug leaving group is a moiety removed by cellular esterases.
 13. Asubstantially pure cyclic purine dinucleotide composition according toclaim 12, wherein the prodrug leaving group is a C6 to C18 fatty acidester.
 14. A substantially pure cyclic purine dinucleotide compositionaccording to claim 1, wherein X₁ and X₂ are both S.
 15. A substantiallypure cyclic purine dinucleotide composition according to claim 14,wherein the cyclic purine dinucleotides present in the compositioncomprise one or more substantially pure Sp,Sp, Rp,Rp, SpRp, or Rp,Spstereoisomers.
 16. A substantially pure cyclic purine dinucleotidecomposition according to claim 1, wherein R1 and R5 are independentlyselected from the group consisting of adenine, guanine, inosine, andxanthine.
 17. A substantially pure cyclic purine dinucleotidecomposition according to claim 16, wherein one or both of R1 and R5 areadenine.
 18. A substantially pure cyclic purine dinucleotide compositionaccording to claim 16, wherein one or both of R1 and R5 are guanine. 19.A substantially pure cyclic purine dinucleotide composition according toclaim 16, wherein R1 is adenine and R5 is guanine
 20. A substantiallypure cyclic purine dinucleotide composition according to claim 1,wherein the composition inhibits STING-dependent type I Interferonproduction at least 2-fold, and more preferably 5-fold or 10-fold, ascompared to c-di-GMP having 3′-5′ linkages.
 21. A substantially purecyclic purine dinucleotide composition according to claim 1, wherein thecyclic purine dinucleotide is formulated with a delivery vehicle whichenhances cellular uptake and/or stability of the cyclic purinedinucleotide.
 22. A substantially pure cyclic purine dinucleotidecomposition according to claim 21, wherein the delivery vehiclecomprises one or more agents selected from the group consisting oflipids, interbilayer crosslinked multilamellar vesicles, biodegradeablepoly(D,L-lactic-co-glycolic acid) [PLGA]-based or poly anhydride-basednanoparticles or microparticles, and nanoporous particle-supported lipidbilayers.
 23. A method of inhibiting an immune response in anindividual, comprising: administering a composition according to claim 1to the individual.
 24. A method of inhibiting STING-dependent type IInterferon production in an individual, comprising: administering acomposition according to claim 1 to the individual in an amountsufficient to inhibit STING-dependent type I Interferon production. 25.A method according to claim 24, wherein the administration isparenteral.
 26. A method according to claim 25, wherein theadministration is subcutaneous, intramuscular, or intradermal.
 27. Amethod according to claim 24, wherein X₁ and X₂ of the cyclic purinedinucleotides present in the composition are both S.
 28. A methodaccording to claim 27, wherein the cyclic purine dinucleotides presentin the composition comprise one or more substantially pure Sp,Sp, Rp,Rp,SpRp, or Rp,Sp stereoisomers.
 29. A method according to claim 28,wherein the cyclic purine dinucleotides present in the compositioncomprise a substantially pure Rp,Rp stereoisomer.